Step 4. Analyze data and call alleles
In this final step, a scatter plot is generated from the normalized allelic discrimination data and alleles are called to determine the genotype of each sample. The inclusion of samples with <5% functional template may interfere with the AutoCall feature of the Sequence Detection Systems (SDS) Software. If this occurs, the alleles can be called manually. The allelic discrimination plot of a typical experiment contains four separate, distinct clusters which represent the no-template controls and three possible genotypes.
>> Recommendation: Collect data by post-plate read on the 7900HT Fast Real-Time PCR System, and use the AutoCall feature of the SDS software to accurately and reproducibly call alleles.
After amplification, the reactions were evaluated via SDS 2.2.1 software using the post-plate read and AutoCalling (quality value threshold: 95) features.
The reproducibility of our recommended SNP genotyping procedure is demonstrated in Figure 3, which shows typical replicate allelic discrimination plots.
The reliability of this procedure is indicated by the results for 10 different TaqMan SNP genotyping assays, which are summarized in Figure 4. For samples containing at least 5% functional template, there is high correlation between observed and expected genotype frequencies. When samples containing less than 5% functional template are included in the analysis, the percent of samples that can be genotyped decreases. Taken together, these data suggest that to ensure that the AutoCall assigns a genotype to a sample, it is important to use only DNA samples that contain >5% functional template as determined by RNase P detection. Otherwise, all samples should be assayed and alleles called manually if the AutoCall feature is unable do so.
Figure 3. Achieve Highly Reproducible and Reliable Data from TaqMan SNP Genotyping Assay. 1 ng, as quantified by RNase P detection, of each human FFPE DNA sample (n=105) was amplified in replicate for 60 cycles followed by AutoCalling using SDS 2.2.1 Software. Gene Symbol: GABRA2, Assay ID: C___8263011_10
Figure 4. Results Summary of TaqMan SNP Genotyping Assays. Data was tabulated after removing all FFPE DNA samples with a ≤ 5% functional template. The “Expected Genotype Frequencies” (columns 9–11) are based on the gene frequencies of each polymorphism in the sample population. The observed genotype frequencies (columns 5–7) fit the expected genotype frequencies within a 95% confidence interval, using an appropriate chi-square test. Finally, the percent of samples genotyped is shown both before and after removal of the low quality samples (columns 12 and 13).